The invention relates to the ergodic (thermal) fragmentation of analyte ions, particularly biopolymer ions, in order to determine their structure and polymer sequences in RF ion traps. Mass spectrometry always means ion spectrometry; hence the term “mass” never refers to the “physical mass” m but always to the “charge-related mass” m/z, where z is the number of excess elementary charges on the ion, i.e. the number of excess protons or electrons. The number z is always understood to be a pure number. The charge-related mass m/z represents the proportion of mass per elementary charge of the ion. The charge-related mass m/z is frequently (somewhat inappropriately) called mass-to-charge ratio, although it is the ratio of mass to the dimensionless number of elementary charges. Whenever the “mass of the ions” is referred to below, it is always to be understood as the charge-related mass m/z, unless expressly stated otherwise.
For an analysis of analyte ions in ion traps, particularly of polymers with sequences of different building blocks such as the biopolymers, the ionization is usually carried out by electrospraying. Electrospray ionization generates hardly any fragment ions; the positive ions are mostly those of the protonated analyte molecule. With electrospray ionization, multiply charged ions of the molecules are usually produced by multiple protonation; this multiple protonation makes the ions heavier than the neutral molecules by a corresponding number of daltons, and are therefore often called “pseudomolecular ions”. For example, doubly and triply charged pseudomolecular ions occur for smaller molecules such as peptides, and ions with up to ten and even a hundred or more charges occur for proteins in the region of physical molecular masses between 5 and 100 kilodaltons.
In order to obtain information on the sequence of polymer building blocks when polymers are the analyte substances, one has to isolate the polymer ions from other ions in the mass spectrometer and then fragment them to produce neutral fragments and charged fragment ions. The mass spectra of the fragment ions are called fragment ion spectra. They contain ion signals arranged like ladders, and the distances between these ion signals allow the type of polymer building blocks and their sequence to be determined. Wherever possible, one starts with doubly to quadruply charged analyte ions for the fragmentation, because these have a very high yield of fragment ions and provide fragment ion spectra which are very easy to interpret.
The spectra of the fragment ions are also called “daughter ion spectra” of the selected “parent ions”. “Granddaughter spectra” can also be measured as fragment ion spectra of selected daughter ions. These daughter ion spectra (and granddaughter ion spectra) can be used to identify structures of the fragmented parent ions; in the case of analyte ions of proteins, for example, it is possible (although difficult for some fragmentation methods) to determine at least parts of the amino acid sequence of a peptide or protein from these spectra.
The analyte substances investigated can belong to different classes of substance, such as proteins, polysaccharides, and also others such as our genetic material DNA. In the following, the invention is described using biopolymer ions, particularly protein ions, without intending to limit the invention to this class of substances. Short-chain proteins with less than about 20 amino acids are usually called “peptides”; when proteins are mentioned here, peptides shall always be included.
Paul RF ion traps use inhomogeneous RF fields to trap the ions. The RF fields generate so-called (fictitious) pseudopotentials, which form a storage well in which both positive and negative ions can be confined. In three-dimensional RF ion traps, the pseudopotential increases in all three spatial directions; in two-dimensional RF ion traps, only in two spatial directions; in the third spatial direction the ions must be trapped by other means, usually by DC potentials.
In the potential wells of RF ion traps, the ions can execute so-called secular oscillations (in addition to the fundamental oscillations imposed by the RF fields), their oscillation frequency decreasing monotonically with increasing charge-related mass m/z. Charging the trap with collision gas damps the secular oscillations; the ions then collect as a cloud in the minimum of the potential well. The specialist is aware of methods for RF ion traps which can be used to analyze the stored ions according to their mass by means of mass-selective ejection. These ion trap mass spectrometers are very inexpensive for the performance they achieve; their use is therefore extraordinarily widespread, with many thousands of these instruments in service. As explained below in more detail, RF ion traps can be designed as so-called 2D ion traps or 3D ion traps. However, the RF ion traps incorporated into mass spectrometers do not have to be used for measuring the mass; the ions stored can be transferred to a different type of mass analyzer for the acquisition of mass spectra.
Mass spectrometers with RF ion traps have characteristics which make them of interest for use in many types of analysis. In particular, selected ion species (the parent ions) can be isolated in the ion trap and then fragmented by various methods. The expression “isolation of an ion species” means that all ion species that are not of interest are removed from the ion trap by means of strong resonant excitation of their secular oscillations or other measures, so that only the required ions, the “parent ions”, remain. These can then be fragmented and form the starting point for the measurement of fragment ion spectra uncontaminated by fragment ions of other substances.
RF ion traps have a peculiarity that is sometimes disadvantageous. They have a “minimum mass threshold” for the storage of ions. Ions with a charge-related mass m/z lower than this mass threshold cannot be stored in the ion trap. These light ions can be accelerated in just a single half-wave of the RF voltage to such an extent that they collide with the electrodes and are thus destroyed. The minimum mass threshold increases in strict proportion when the RF voltage is increased.
When, in the following, RF ion traps are mentioned, this refers not only to the ion traps in ion trap mass spectrometers but, in general, all ion traps in which ions are stored by pseudopotentials created by RF fields, and any gaps in the envelope of the pseudopotentials are closed by other means such as DC potential gradients. These ion traps also include hexapole and octopole rod systems, for example, and also axial arrangements of ring diaphragms with alternately applied phases of the RF.
Two fundamentally different types of fragmentation are now available in the various types of ion trap: “ergodic” fragmentation and “electron-induced” fragmentation. These two types of fragmentation lead to two significantly different types of fragment ion spectra, whose information content is complementary and which lead to particularly detailed information on the structures of the analyte ions when both types of fragment ion spectra are measured.
The term “ergodic” fragmentation of analyte ions here means a fragmentation where a sufficiently large excess of internal energy in the analyte ions leads to fragmentation. The excess energy can, for example, be introduced by a large number of relatively gentle collisions of the analyte ions with a collision gas; or by the absorption of a large number of photons from an infrared radiation source.
According to the ergodic theorem originally formulated by Boltzmann as a hypothesis, in a closed system such as a complex molecular analyte ion, when a certain energy is present, every state which can be realized with this energy will actually be realized in the course of time. This ergodic theorem is a mathematically proven form of the ergodic hypothesis, more precise for the ergodic quasi-hypothesis, in which every state will be realized in any pre-chosen approximation. Since fragmentation produces a possible, even if an irreversible, state, namely the creation of two particles from the analyte ion, fragmentation will occur at some stage. By absorbing energy, analyte ions called “metastable” ions are temporarily created, which then decompose at some time. The decomposition itself is characterized by a “half life”, which is, however, dependent on the amount of surplus energy and cannot be determined unambiguously by today's methods.
The probability of ergodic cleavage of a certain bond depends on its binding energy. Only the weakest bonds of the analyte ion have a high probability of being cleaved. In proteins, the weakest bonds (except for side chains) are the so-called peptide bonds between the amino acids, which lead to fragments of the b and the y series, some occurring as charged fragment ions, some as neutral particles. Since the peptide bonds between different amino acids have slightly different binding energies, some peptide bonds of the analyte ion are more likely to be cleaved, and others less likely. As a result, not all fragment ions from peptide bonds in the fragment ion spectrum have the same intensity. Non-peptide bonds are so seldom cleaved that their fragments are typically not present in measurable quantities. In ergodic fragment ion spectra, there is no information about side chains like modifications of the amino acids, because all side chains get lost during fragmentation due to their low binding energy.
The conventional type of fragmentation of the analyte ions in RF ion traps is ergodic fragmentation by collisions of the somehow accelerated analyte ions with the collision gas contained in the ion trap, the excess internal energy of moving analyte ions being accumulated by collisions with the stationary collision gas molecules in the ion trap. In order for the collisions to be able to pump any energy into the analyte ion at all, they have to occur with a minimum collision energy. Since even gentle collisions of the analyte ions with the collision gas always may cause an internal cooling, i.e. a loss of internal energy from the analyte ion, there is always competition between “heating” and “cooling”; physically heavy ions, in particular, require a higher collision energy for the heating than light ions.
There are strict limitations on the collision gas in RF ion traps. On the one hand, the collision gas should serve to dissipate the kinetic energy of the analyte ions in order to collect the ions in the center of the ion trap. Here it is advantageous to use a collision gas that has small molecules and a relatively high density in the order of 10−1 to 10−2 pascal. Helium is usually used as the collision gas. Under these conditions, the mass-selective ejection of the ions for measurement of the masses is not significantly disturbed. This small-molecule collision gas is not particularly well suited to collision-induced dissociation. Nevertheless, no other collision gases have become established in commercial mass spectrometers.
In three-dimensional RF ion traps, the collision energy is generated in the conventional way by a limited resonant excitation of the secular ion oscillations of the parent ions with a dipolar alternating voltage. This leads to many collisions with the collision gas without removing the ions from the ion trap. The ions can accumulate energy in the collisions, which finally leads to ergodic decomposition and the creation of fragment ions. Until a few years ago, this collision-induced dissociation (CID) was the only known type of fragmentation in ion traps.
This collision-induced dissociation in three-dimensional RF ion traps also has disadvantages, however. For physically heavy analyte ions, it is necessary to set the RF voltage for storing the ions at a very high level in order to produce sufficiently hard collision conditions. This results in a very high minimum mass threshold for the ion trap. Ions below this mass threshold can no longer be stored; they are lost. The fragment ion spectrum therefore only starts at a mass which is about one third of the charge-related mass m/z of the analyte ion; the fragment ion spectrum can no longer provide any information on the lighter fragment ions because these ions are lost. Multiply charged physically heavy analyte ions of m>3000 dalton regularly have a low charge-related mass m/z of only about 800 to 1400 daltons, owing to the large number of protons; these analyte ions cannot be fragmented at all because the RF voltage cannot be set high enough to produce sufficient numbers of high-energy collisions.
Two-dimensional ion traps that are used as mass analyzers always have the form of quadrupole rod systems. In these 2D ion traps, the ergodic fragmentation is usually carried out in the same way by resonant excitation of the secular oscillations of the analyte ions for collisions with the collision gas; they therefore have the same problems as three-dimensional ion traps. In two-dimensional ion traps that are not also used as mass analyzers, the ion traps can take the form of hexapole or octopole rod systems, for example. In this case, the analyte ions can be injected axially into the collision gas with a specified kinetic energy. Here, also, the internal energy of the analyte ions is increased by a large number of collisions, and many analyte ions which have become metastable are subsequently ergodically fragmented. But here there are also upper limits for the mass of the analyte ions which can be fragmented, and here as well there are minimum mass thresholds below which fragment ions cannot be collected.
In order to also store very small fragment ions (particularly the so-called immonium ions, which consist of only one amino acid and are produced by internal fragmentation of fragment ions) by collision-induced dissociation, special methods have recently been elucidated which make use of the slow, metastable decomposition of the ions by the ergodic fragmentation process. These methods are quite useful, if a little complicated; they do not, however, deliver true-to-quantity reproduction of the fragment ions in the fragment ion spectra.
There remains the big disadvantage of collision-induced dissociation that with physically heavier analyte molecules above about 3000 daltons, the corresponding analyte ions can hardly be fragmented at all.
Document WO 02/101 787 A1 (S. A. Hofstadler, and J. J. Drader) elucidates that infrared multiphoton dissociation (IRMPD) can also be used in RF ion traps. The infrared radiation here is introduced into a three-dimensional RF ion trap via an evacuated hollow fiber with an optically reflective internal coating, through the perforated ring electrode. Thus, a further method for ergodic fragmentation is available with RF ion traps. This type of fragmentation is very advantageous because it can be carried out at low RF voltages; the small fragment ions are then also stored. The internal surfaces of the ion trap must be kept extremely clean because any molecules adhering to the walls are detached by the irradiation of the infrared photons, and these molecules then react in a variety of ways with the stored analyte ions. This is the main reason why there are still no commercially available ion trap mass spectrometers with this type of fragmentation.
We now turn to the electron-induced fragmentation methods. About ten years ago, a completely new type of fragmentation of protein ions was discovered: a non-ergodic fragmentation induced by the capture of low-energy electrons (ECD=“electron capture dissociation”). By the direct neutralization of an associated proton, which is then lost as a radical hydrogen atom, the potential equilibrium of the protein ion in the vicinity of the neutralized proton is disturbed so much that a cleavage of the amino acid chain is induced by corresponding rearrangements. The cleavage fragmentation does not concern the peptide bonds, but adjacent bonds, leading to so-called c- and z-fragment ions.
This type of fragmentation is particularly easy to carry out in ion cyclotron resonance mass spectrometers because the low-energy electrons from a thermion cathode can easily be supplied along the lines of magnetic force to the stored cloud of analyte ions. ECD fragmentation can only be used in RF ion traps with some difficulty because the strong RF fields do not easily allow the low-energy electrons to come very close to the cloud of analyte ions. Nevertheless, there are a number of different solutions for ECD fragmentation in RF ion traps, but they each require costly apparatus and have not yet achieved a satisfactory sensitivity.
A method for the fragmentation of ions in RF ion traps has recently been elucidated which produces fragmentations similar to electron capture dissociation (ECD) but by a different reaction: “electron transfer dissociation” (ETD). This can easily be carried out in ion traps by adding suitable negative ions to the stored analyte ions. Methods of this type have been described in the patent publications DE 10 2005 004 324.0 (R. Hartmer and A. Brekenfeld) and US 2005/0199804 A1 (D. F. Hunt et al.). The fragment ions here belong (as in ECD) to the so-called c and z series, and are therefore very different to the fragment ions of the b and y series obtained by ergodic fragmentation. The fragments of the c and z series have significant advantages for determining the amino acid sequence from the mass spectrometric data, not least because the ETD fragment ion spectra can extend to lower masses than the collisionally induced fragment ion spectra. In particular, all the side chains are preserved during electron transfer dissociation, including the important posttranslational modifications such as phosphorylations, sulfations und glycosylations.
The fragmentation of protein ions by electron transfer (ETD) in an RF ion trap is brought about in a very simple way by reactions between multiply charged positive protein ions and suitable negative ions. Suitable negative ions are usually polyaromatic radical anions, such as those of fluoranthene, fluorenone, anthracene or other polyaromatic compounds. With radical anions, the chemical valences are not saturated, so they can easily donate electrons in order to achieve an energetically advantageous non-radical form. They are generated in NCI ion sources (NCI=“negative chemical ionization”), most probably by single electron capture or by electron transfer. NCI ion sources are constructed, in principle, like ion sources for chemical ionization (CI ion sources), but operated differently in order to obtain large quantities of low-energy electrons. NCI ion sources are also called electron attachment ion sources.
It is now known that an electron transfer from highly excited neutral particles, for example by highly excited helium atoms from a “fast atom bombardment” (FAB) particle source, can also take place (DE 10 2005 005 743 A1, R. Zubarev et al.). This type of fragmentation is abbreviated to MAID (“metastable-atom induced dissociation”). Here, also, there are ECD-type fragment ion spectra. For the non-ergodic fragmentation process by neutralization of a proton by an electron, the source of the electron seems to be unimportant. The ECD, ETD and MAID types of fragmentation can therefore all be collectively referred to as “electron-induced” types of fragmentation.
Evaluation of the fragment ion spectra is very simple if they were produced from doubly to about quadruply charged parent ions, because doubly to quadruply charged fragment ions can be identified from the mass separations of their isotopic pattern, and because the fragment ion spectra are not too complex. It is a different situation if highly charged parent ions, for example parent ions carrying ten to thirty charges are subjected to this fragmentation process. The number of different fragment ions is extraordinarily high, and the vast majority of fragment ions cluster in the region of charge-related masses m/z from about 600 to 1200 daltons. The fragment ion spectrum is so complex that an evaluation is hardly possible, especially since the isotope patterns can no longer be mass-resolved in RF ion trap mass analyzers, and therefore the charge level can no longer be determined.
For de-novo sequencing, and also for other purposes such as the determination of posttranslational modifications (PTM), it is particularly advantageous to evaluate spectra acquired both by ergodic fragmentation and by electron-induced dissociation.
Ergodic fragmentation initially cleaves all posttranslational modifications that are only weakly bonded, such as phosphorylations, sulfations and glycosylations, and essentially displays the naked sequence of the unmodified amino acids of the analyte ions. Therefore, the types and positions of the posttranslational modifications cannot be identified. In contrast, these modification groups are not cleaved off by electron-induced fragmentation. In comparison with the ergodically obtained fragment ion spectra, an additional mass at an amino acid thus shows both the type and also the position of the modification. These extraordinarily important investigative results can only be obtained by comparing both types of fragment ion spectra.
De-novo sequencing is always desirable when searching in a protein sequence database using a search engine has not provided any useful results, for example because a protein of this type is not yet available in the database. A comparison of ergodic and electron-induced fragment ion spectra allows the ion signals to be immediately assigned to the c/b series or z/y series because there is a fixed mass difference between c-ions and b-ions and also between z-ions and y-ions, which makes identification easy. It is therefore very easy to read out partial sequences for both series of fragment ions.
The simple generation of ETD fragment ion spectra thus does not obviate the need to generate extensive ergodic fragment ion spectra, since it is only with both kinds of fragment ion spectra in parallel that a lot of valuable information on the structure of the analyte ions can be obtained. As has been described in detail above, the acquisition of informative ergodic fragment ion spectra from heavier analyte ions is still extraordinarily difficult, if not impossible, at present.